New species of Yamadazyma from rotting wood in China

Abstract Yamadazyma is one of the largest genera in the family Debaryomycetaceae (Saccharomycetales, Saccharomycetes) with species mainly found in rotting wood, insects and their resulting frass, but also recovered from flowers, leaves, fruits, tree bark, mushrooms, sea water, minerals, and the atmosphere. In the present study, several strains obtained from rotting wood in Henan and Yunnan Provinces of China were isolated. Based on morphology and a molecular phylogeny of the rDNA internal transcribed spacer region (ITS) and the D1/D2 domain of the large subunit (LSU) rDNA, these strains were identified as three new species: Yamadazymaluoyangensis, Y.ovata and Y.paraaseri; and three previously described species, Y.insectorum, Y.akitaensis, and Y.olivae. The three new species are illustrated and their morphology and phylogenetic relationships with other Yamadazyma species are discussed. Our results indicate a high undiscovered diversity of Yamadazyma spp. inhabiting rotting wood in China.

Yamadazyma species can be originally found in tropical, subtropical, and temperate regions of different continents, but most known species appear to exist in Asia and South America Groenewald et al. 2011;Kurtzman 2011;Lachance et al. 2011;Kaewwichian et al. 2013;Junyapate et al. 2014;Jindamorakot et al. 2015;Lopes et al. 2015;Wang et al. 2015;Burgaud et al. 2016;Khunnamwong and Limtong 2016;Nagatsuka et al. 2016). The genus has been heavily studied in Asia, and 17 species of Yamadazyma were previously reported in Japan and Thailand Groenewald et al. 2011;Kurtzman 2011;Lachance et al. 2011;Kaewwichian et al. 2013;Junyapate et al. 2014;Jindamorakot et al. 2015;Wang et al. 2015;Khunnamwong and Limtong 2016;Nagatsuka et al. 2016). By contrast, little is known about Yamadazyma spp. in China. To date, only three Yamadazyma species have been described in China, namely C. diospyri, Y. dushanensis, and Y. paraphyllophila (Lachance et al. 2011;Kaewwichian et al. 2013;Wang et al. 2015). In this study, we collected rotting wood samples from Yunnan and Henan Provinces in China. After isolation and examination, three new species and three known species of Yamadazyma were identified based on phenotypic characteristics and phylogenetic analysis, increasing the species diversity of Yamadazyma in China.

Sample collection and yeast isolation
Samples of rotting wood were collected in the Xishuangbanna Primeval Forest Park (Yunnan Province, China) and the Tianchi Mountain National Forest Park (Henan Province, China). The Xishuangbanna Primeval Forest Park (21°98'N, 100°88'E) is 500 m above sea level (MASL), with a hot and humid climate. The average annual temperature is between 16 °C and 28 °C, and the average annual rainfall is above 1,100 mm. The Tianchi Mountain National Forest Park (34°33'N, 112°28'E) is at 850 MASL, with a continental monsoon climate, average annual temperature of 14-16 °C, and average annual rainfall between 800 mm and 900 mm. Fifty decayed wood samples were collected during July and August in 2018-2020. The samples were stored in sterile plastic bags and transported under refrigeration to the laboratory over a period of no more than 24 h. The yeast strains were isolated from rotting wood samples in accordance with the methods described by Wang et al. (2015). Each sample (1 g) was added to 20 ml sterile yeast extract-malt extract (YM) broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, pH 5.0 ± 0.2) supplemented with 0.025% sodium propionate and 200 mg/L chloramphenicol in a 150 ml Erlenmeyer flask and then cultured for 3-10 days on a rotary shaker. Subsequently, 0.1 ml aliquots of the enrichment culture and appropriate decimal dilutions were spread on YM agar plates and then incubated at 25 °C for 3-4 days. Different yeast colony morphotypes were then isolated by repeated plating on YM agar and then stored on YM agar slants at 4 °C or in 15% glycerol at -80 °C.

Phenotypic study
Morphological and physiological properties were determined according to those used by Kurtzman et al. (2011). The beginning of the sexual stage was determined by incubating single or mixed cultures of each of the two strains on cornmeal (CM) agar, 5% malt extract (ME) agar, dilute (1:9) V8 agar, or yeast carbon base plus 0.01% ammonium sulfate (YCBAS) agar at 15 and 25 °C for 6 weeks (Kurtzman 2011;Wang et al. 2015). The assimilation of carbon and nitrogen compounds and related growth requirements were tested at 25 °C. The effects of temperature from 25-40 °C were examined in liquid and agar plate cultures. Photomicrographs were taken using a Leica DM 2500 microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a Leica DFC295 digital microscope color camera, with bright field, phase contrast, and DIC optics. Novel taxonomic descriptions and proposed names were deposited in Myco-Bank (http://www.mycobank.org; 8 June 2021) (Crous et al. 2004).

DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from the yeast using an Ezup Column Yeast Genomic DNA Purification Kit, according to the manufacturer's instructions (Sangon Biotech, Shanghai, China). The internal transcribed spacer (ITS) and the D1/D2 domain of the large subunit (LSU) rDNA were respectively amplified using ITS5/ITS4 (White et al. 1990) and NL1/NL4 (Kurtzman and Robnett 1998) primers with cycling conditions of 94 °C/30 s, 55 °C/50 s, 72 °C/60 s. All the PCR protocols had 35 cycles including 94 °C/5 min initial denaturation and 72 °C/10 min final extension.

Phylogenetic analysis
The sequences obtained in this study and the reference sequences downloaded from GenBank (Table 1) were aligned using MAFFT v 7(https://mafft.cbrc.jp/alignment/ server/large.html;) (Katoh et al. 2019) and manually edited using MEGA7 (Kumar et al. 2016). The best-fit nucleotide substitution models for individual and combined datasets were selected using jModelTest v2.1.7 (Darriba et al. 2012) according to the Akaike information criterion. Phylogenetic analyses of combined gene regions (ITS and D1/D2 LSU) were performed using MEGA7 for maximum parsimony (MP) analysis (Kumar et al. 2016) and PhyML v3.0 for maximum likelihood (ML) analysis (Guindon et al. 2010). Scheffersomyces coipomoensis (CBS 8178) and Babjeviella inositovora (CBS 8006) were used as the outgroup taxa based on Haase et al. (2017) and Nagatsuka et al. (2016).  MP analysis was run using a heuristic search option of 1,000 search replicates with random-addition of sequences and tree bisection and reconnection (TBR) as the branch-swapping algorithm. Gaps were treated as missing data. Bootstrapping with 1,000 replicates was performed to determine branch support (Felsenstein 1985). Parsimony scores of tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC) were calculated for each generated tree. ML analysis was performed using a GTR site substitution model, including a gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). Branch support was evaluated using a bootstrapping method of 1,000 bootstrap replicates (Hillis and Bull 1993). The phylogenies from MP and ML analyses were displayed using Mega7 and FigTree v1.4.3 (Rambaut 2016), respectively. ML and MP bootstrap support values above 50% are shown as first and second positionS above nodes, respectively.

Molecular phylogeny
The alignment based on the combined nuclear dataset (ITS and D1/D2 LSU) included 65 taxa and two outgroup taxa (Scheffersomyces coipomoensis and Babjeviella inositovora), and was comprised of 1,103 characters including gaps (576 for ITS and 527 for D1/D2 LSU) in the aligned matrix. Of these characters, 351 were constant, 455 variable characters were parsimony-uninformative, and 297 characters were parsimony-  informative. The heuristic search using MP analysis generated the most parsimonious tree (TL = 979, CI = 0.297, RI = 0.653, RC = 0.248). The best model applied in the ML analysis was GTR+I+G. The ML analysis yielded a best scoring tree with a final optimization likelihood value of -11,006.61. Both methods for phylogenetic tree inference resulted in a similar topology. Therefore, only the best scoring PhyML tree is shown with BS and BT values simultaneously in Figure 1. According to the phylogenetic tree (Figure 1), three known species, Y. insectorum, Y. akitaensis, and Y. olivae, were part of Yamadazyma. Yamadazyma luoyangensis, Y. ovata, and Y. paraaseri are new to science based on the distinct and well-supported molecular phylogenetic placement and morphological differences with their closest described relatives (Table 2). Phylogenetically, Y. luoyangensis clustered together with Y. ovata and other species, including Y. mexicana, Y. terventina, Y. dushanensis, and C. trypodendroni, while Y. paraaseri was closely related to C. aaseri with high bootstrap support (98% ML/98% MP).  Etymology. The species name luoyangensis refers to the geographical origin of the type strain of this species.
Description. The cells are ovoid to ellipsoid (2-4 × 3.5-7 µm) and occur singly or in pairs after being placed in YM broth for three days at 25 °C ( Figure 2A). Budding is multilateral. After three days of growth on YM agar at 25 °C, the colonies are white to cream-colored, buttery, and smooth, with entire margins. After seven days at 25 °C on a Dalmau plate culture with CM agar, pseudohyphae are formed, but true hyphae are not ( Figure 2B). Asci or signs of conjugation are not observed on sporulation media. Glucose, galactose, trehalose, and cellobiose are fermented, but maltose, sucrose, melibiose, lactose, melezitose, raffinose, d-xylose, and inulin are not. Glucose,galactose,sucrose,maltose,trehalose,cellobiose,salicin,arbutin,melezitose,inulin,glycerol,erythritol,ribitol,galactitol,succinate,citrate,and ethanol are assimilated. No growth is observed in l-sorbose, melibiose, lactose, raffinose, myo-inositol, 2-keto-d-gluconate, d-glucuronate, dllactate, or methanol. In nitrogen-assimilation tests, growth is present on ethylamine, l-lysine, glucosamine, and d-tryptophan, while growth is absent on nitrate, nitrite, cadaverine, creatine, creatinine, and imidazole. Growth is observed at 35 °C but not at 37 °C. Growth in the presence of 10% NaCl with 5% glucose is present, but growth in the presence of 0.01% cycloheximide and 1% acetic acid is absent. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.    Notes. Two isolates representing Y. luoyangensis were resolved in a well-supported clade and are most closely related to Y. mexicana (Figure 1). Yamadazyma luoyangensis can be distinguished from Y. mexicana based on ITS and D1/D2 LSU loci (4/592 in ITS and 10/531 in D1/D2 LSU). Physiologically, Y. luoyangensis differs from Y. mexicana by its ability to assimilate inulin and 5-keto-d-gluconate and its inability to assimilate lactose, raffinose, and 2-keto-d-gluconate. Additionally, Y. mexicana grows at 37 °C, while Y. luoyangensis does not (Table 2) (Kurtzman 2011).  Etymology. The species name ovata refers to the ovoid cell morphology of the type strain.

Yamadazyma ovata
Description. The cells are ovoid to ellipsoid (2-3 × 3-6.5 µm) and occur singly or in pairs after growth in a YM broth for three days at 25 °C ( Figure 3A). Budding is multilateral. After three days of growth on YM agar at 25 °C, the colonies are white to cream-colored, buttery, and smooth with entire margins. After nine days at 25 °C, on a Dalmau plate culture with CM agar, pseudohyphae consisting of elongated cells with lateral buds are formed ( Figure 3B). True hyphae are not observed. Asci or signs of conjugation are not observed on sporulation media. Glucose, galactose, and trehalose are fermented, but maltose, sucrose, melibiose, lactose, cellobiose, melezitose, raffinose, d-xylose, and inulin are not. Glucose,galactose,sucrose,maltose,trehalose,cellobiose,salicin,melibiose,melezitose,glycerol,erythritol,ribitol,xylitol,, 5-lactone, 2-keto-d-gluconate, d-gluconate, succinate, citrate, and ethanol are assimilated. No growth is observed in l-rhamnose, lactose, raffinose, inulin, myo-inositol, d-glucuronate, dl-lactate, or methanol. In nitrogen-assimilation tests, growth is present on l-lysine, creatine, glucosamine, and d-tryptophan, while growth is absent on nitrate, nitrite, ethylamine, cadaverine, creatinine, or imidazole. Growth is observed at 37 °C, but not at 40 °C. Growth in the presence of 16% NaCl with 5% glucose is present, but growth in the presence of 0.01% cycloheximide and 1% acetic acid is absent. Starch-like compounds are not produced. Urease activity and diazonium blue B reactions are negative.
Notes. Two strains representing Y. paraaseri were clustered in a well-supported clade and were phylogenetically related to C. aaseri [7]. Yamadazyma paraaseri can be distinguished from C. aaseri based on ITS and D1/D2 LSU loci (8/573 in ITS and 8/531 in D1/D2 LSU). Physiologically, the ability to assimilate d-glucosamine and inulin and the inability to assimilate xylitol and d-glucono-1, 5-lactone are the primary differences between Y. paraaseri and its closest relative, C. aaseri. Additionally, C. aaseri can grow in 10% NaCl with 5% glucose, while Y. paraaseri cannot (Table 2) (Lachance et al. 2011).

Discussion
In this work, six Yamadazyma species were identified based on morphology and molecular phylogeny. All species were isolated from rotting wood collected in Henan and Yunnan Provinces, China. Yamadazyma luoyangensis, Y. ovata, and Y. paraaseri are proposed as new species in Yamadazyma due to their well-supported phylogenic positions and distinctive physiological traits. Also, three known species of Yamadazyma, Y. insectorum, Y. akitaensis, and Y. olivae, were clearly identified by both morphological and molecular approaches.
In the past, methods of species identification of Yamadazyma were based only on morphology and physiological characters such as the shape of ascospores and reactions in standard growth and fermentation tests (Billon-Grand 1989). Recent molecular phylogenetic analyses demonstrate that determining species boundaries using only morphology and physiological characters is not possible due to their variability under changing environmental conditions (Kurtzman 2011;Lachance et al. 2011). D1/D2 LSU sequence is an appropriate marker to identify species of Yamadazyma species through phylogenetic analysis, as revealed by Kurtzman and Robnett (1998). Many Yamadazyma species are described based on a polyphasic approach together with morphological and physiological characterization (Suh et al. 2005;Kurtzman 2007;Imanishi et al. 2008;Nagatsuka et al. 2009;Am-In et al. 2011). However, none to only two substitutions are present in D1/D2 LSU sequences of the ex-type strains of the closest related species within Yamadazyma, such as C. diddensiae and C. naeodendra, Y. akitaensis and Y. nakazawae as well as C. jaroonii and C. songkhlaensis (Groenewald et al. 2011;Wang et al. 2015). The ITS sequences show more variation between these closely related well-defined species in contrast to the low nucleotide differences in D1/D2 LSU sequences (Groenewald et al. 2011). Although D1/D2 LSU sequence is still an appropriate region to use for higher level taxon delimitations, it is clear that this sequence alone is not sufficient for species delimitation in the Yamadazyma clade. The ITS sequence is thus a good additional marker to obtain a better understanding of relatedness among Yamadazyma species.
Yamadazyma species have a worldwide distribution and are isolated from diverse substrates. They can be found in flowers, leaves, fruits, tree bark, mushrooms, sea water, mineral and atmosphere, but most known species appear to exist in rotting wood, insects and their resulting frass (Groenewald et al. 2011;Kurtzman 2011). This clade also includes the clinically significant species C. aaseri, C. conglobata, C. pseudoaaseri, and Y. triangularis (Kurtzman 2011;Lachance et al. 2011). These studies expanded our knowledge on the substrates where Yamadazyma species can occur, but on the other hand demonstrated the complicated ecological function of this genus. In this study, three known species and three new species were identified from rotting wood in China. Further research will focus on Yamadazyma diversity from a wide range of substrates.